Dissertation zur Erlangung des Doktorgrades der Fakultät für Chemie und Pharmazie der Ludwig-Maximilians-Universität München RNA-dependent chromatin association of transcription elongation factors and Pol II CTD kinases Sofia Luciana Battaglia aus San Pedro, Argentinien 2017 2 Erklärung Diese Dissertation wurde im Sinne von § 7 der Promotionsordnung vom 28. November 2011 von Herrn Prof. Dr. Patrick Cramer betreut. Eidesstattliche Versicherung Diese Dissertation wurde eigenständig und ohne unerlaubte Hilfe erarbeitet. München, den 07.04.2017 ---------------------------- Sofia Battaglia Dissertation eingereicht am 12.04.2017 1. Gutachter: Prof. Dr. Patrick Cramer 2. Gutachter: PD Dr. Dietmar Martin Mündliche Prüfung am 29.05.2017 3 4 Acknowledgments First of all, I would like to thank my supervisor Professor Patrick Cramer for giving me the opportunity to work in his group. He gave me freedom to explore the field, but was also present for discussion and troubleshooting any time when needed. I thank him for all the support, advice and positivity during the last four years. His trust was essential for my development as a scientist. I am very thankful to have worked with Michael Lidschreiber, who was an amazing collaborator. He is a smart co-worker and a good friend now. He was very calm even when the science was not doing what we expected. Finally, we managed to have a super nice story! I thank Margaux Michel for troubleshooting together during the optimization of the ChIP protocol and many other general discussions. I also thank her for her wonderful atmosphere. Seychelle Vos helped me with one very important and interesting experiment for this thesis. She gave me a lot of smart advices and was always very motivated with our work. I also thank Amelie Schreieck for sharing her expertise in several techniques at the beginning of my PhD. I also want to give Kerstin Maier a big thank you for constant discussions and advise as a desk colleague and an expert in the yeast field. Petra Rus was always very nice and gave our little yeast lab the perfect atmosphere to work in a good mood. She was also very flexible to help me with experiments when needed. Mark Böhning was a perfect join in the lab, we often stayed late discussing about our projects and trying to help one another. I thank Carlo Baejen for teaching me the PAR-CLIP protocol. Christian Roth was very patient showing me how to deal with sequencing data and perform basic analysis myself. In this regard, I also want to thank Johannes Söding for discussions about PAR-CLIP data. I also would like to specially thank Luis Fonseca, Marc Böhning, Anna Sawicka, Kerstin Maier, Michael Lidschreiber and Quinn Klinge for critical reading of parts of this thesis. Special thanks to my committee members PD Dr. Dietmar Martin, Professor Peter Becker, Professor Andreas Ladurner, Dr. Fabiana Perocchi and Professor Klaus Förstemann for their time. And of course a big thanks to all the members of the Cramer group. It was an enormous pleasure to work with such a good team. I deeply thank Luis Fonseca, my best friend. Without his company and support this thesis would have been a lot harder for me. His good mood and positivity were a big help for me in difficult times. It was also very productive and fun having science discussions with him. Many thanks to my dear girl friends for all the great moments together. Spending time with them was a way to recharge my energies. I have been very blessed in my life to have their love and friendship. I thank my siblings, Alba y Francisco, for being part of my family and sharing so many wonderful things in this life. I love laughing with them. And my very special thanks go to my parents, Irene and Horacio, that even being far away they were always close and supportive any day and any time I needed. I thank them for the person who I am today. Gracias. I also thank my lovely Oma. I am sure she would be celebrating this moment with me. 5 6 Summary For transcription through chromatin, RNA polymerase (Pol) II associates with transcription factors. Recent work revealed that transcription factors also interact with the nascent RNA to regulate gene expression. The focus of this thesis relies on the characterization of this form of interactions during the process of transcription elongation. I used our recently optimized PAR-CLIP protocol to show that many elongation factors (EFs) crosslink to RNA emerging from transcribing Pol II in the yeast Saccharomyces cerevisiae. These in vivo direct interactions were most notable for the kinases Ctk1 and Bur1 that phosphorylate the C-terminal repeat domain (CTD) of the largest Pol II subunit and for the histone H3 methyltransferases Set1 and Set2. Bioinformatic analysis indicated that most EFs crosslink preferentially to mRNAs, rather than to unstable non-coding RNAs, consistent with their recruitment to transcribed protein-coding genes. Furthermore, I developed an RNA degradation assay to test whether the observed RNA-protein interactions affect protein-chromatin binding under native conditions. Comparing protein-chromatin binding in the presence and absence of RNA revealed that RNA contributes to chromatin association in particular of the CTD serine 2 kinases Ctk1 and Bur1 and the histone H3 methyltransferases Set1, Set2 and Dot1. Additionally, I confirmed the in vivo observed EF-RNA interactions for an active CTDK-I kinase complex in vitro using fluorescence anisotropy. Finally, I optimized our ChIP protocol for high-throughput sequencing and performed ChIP-Seq experiments of most Pol II EFs and histone marks involved in this study. Comparison of factor occupancies on DNA (ChIP-Seq) and on RNA (PAR-CLIP) revealed that interactions of EFs with nascent RNA are established before EFs are recruited to chromatin. Taken together, these studies argue for a role of nascent RNA in EF recruitment. In this model, EF-RNA interactions facilitate assembly of the elongation complex on transcribed genes when RNA emerges from Pol II, and loss of EF-RNA interactions upon RNA cleavage at the polyadenylation site trigger disassembly of the elongation complex. 7 8 Publications Part of this work has been published or is in the process of being published. 2017 RNA-dependent chromatin association of transcription elongation factors and Pol II CTD kinases Sofia Battaglia*, Michael Lidschreiber*, Carlo Baejen, Phillipp Torkler, Seychelle M. Vos, Patrick Cramer (* joint first authorship). Under revision. Author contributions: SB and CB performed PAR-CLIP experiments. SB and SMV purified recombinant CTDK-I and performed in vitro assays. SB performed ChIP-Seq and chromatin association experiments. ML and PT carried out PAR-CLIP data analysis. ML carried out PAR-CLIP normalization and ChIP-Seq data analysis. PC supervised research. SB, ML and PC wrote the manuscript. 2017 Genome-wide analysis of RNA polymerase II termination at protein-coding genes Carlo Baejen, Jessica Andreani, Phillipp Torkler, Sofia Battaglia, Bjoern Schwalb, Michael Lidschreiber, Kerstin C. Maier, Andrea Boltendahl, Petra Rus, Stephanie Esslinger, Johannes Söding, and Patrick Cramer Molecular Cell Author contributions: CB and PC designed the study. CB planned and coordinated experiments. KCM, SE and PR cloned strains and performed anchor- away and 4tU-Seq experiments. KCM, PR and CB carried out growth rate experiments. AB and CB performed PAR-CLIP experiments. SB performed ChIP-Seq and ChIP-qPCR experiments. KCM performed ChIP-qPCR experiments. JA, PT, BS, JS, CB and PC planned data analyses. JA and BS carried out 4tU-Seq data analyses. JA carried out ChIP-Seq data analyses. PT, CB and ML carried out PAR-CLIP data analyses. JS supervised data analysis and modelling. PC supervised research. CB and PC wrote the manuscript with input from all authors. 2017 Intergenic non-coding transcription utilises an RNA-based mechanism to drive transcriptional interference Beth R. Watts, Sina Wittmann, Sofia Battaglia, Cornelia Kilchert, Dong-hyuk Heo, Patrick Cramer, and Lidia Vasiljeva Under revision. Author contributions: BRW and LV conceived and designed experiments. BRW performed all experiments except: PAR-CLIP (SB), Mmi1 purification (D-HH), analysis of ChIP-seq, RNA-seq and PAR-CLIP analyses (SB and SW). CK and SW helped with generating strains. BRW and LV wrote the paper and all authors edited the manuscript. 9 Additional ongoing collaborations: 2017 The stress responsive kinase Hog1 binds mRNA in response to osmotic stress Research group of Francesc Posas (UPF Departament de Ciències Experimentals i de la Salut, Barcelona, Spain. Manuscript in preparation. My contributions: SB performed conditional PAR-CLIP after salt stress 10 Table of Contents Erklärung ............................................................................................................................. 3 Eidesstattliche Versicherung ......................................................................................... 3 Acknowledgments ............................................................................................................. 5 Summary .............................................................................................................................. 7 Publications ......................................................................................................................... 9 1 Introduction .............................................................................................................. 13 1.1 RNA synthesis by RNA polymerases ......................................................................